Functional Identification Of E3 Ligase GhPUB17 In Cotton Resistance To Verticillium Dahliae

Verticillium dahliea causes serious yield losses in production and quality of cotton fiber as a major disease to cotton growth over the world.Exploration and identification of interaction between cotton and pathogens can provide new views of theory foundation and gene resources for performing cotton molecular disease-resistance breeding.During the process of plants interacting with pathogens,great amount of proteins covering enzymes cooperate each other for insurance of producing and transmitting of disease-resistant signals.The ubiquitin proteasome system(UPS)is a major pathway for degradation and modification of proteins inside eukaryotic organisms,which widely involves in management of most proteins.In recent years,the key component of UPS as E3 ligase was identified as the controller involved in plant resistance to relative pathogens of multiple species.In our study,we identified an E3 ligase involved in cotton resistance to Verticillium dahliae and the achieved results are listed as followings.1.GhPUB17 was identified as a homolog to PUB17 in Arabidopsis thaliana which reported involving in immunoregulation to pathogens.With inoculation of anti-disease related hormones including SA and MeJA to cotton seedlings,the expression level of GhPUB17 was both up-regulated respectively,similar to thw results of inoculation of V.dahliae strain V991.To explore the function of GhPUB17 in cotton resistance to V.dahliae,we constructed different vertors for GhPUB17 to regulate expression quantity of GhPUB17 for investigating its functions.2.With operation of virus induced gene silencing(VIGS)and RNAi silencing for knocking down of GhPUB17,the resistance of cotton seedlings to V.dahliae was enhanced.With overexpression of GhPUB17,cotton resistance to V.dahliae was repressed compared to GhPUB17 down expressed lines and the wildtype.With inoculation of Botrytis cinerea,no differences were observed,indicating that GhPUB17 might manage cotton resistance to V.dahliae specifically.3.The cotton endogenous marker genes of JA and SA hormone pathways were analyzed by qRT-PCR after all GhPUB17 transgenic lines and WT plants were inoculated with V.dahliae.Results displayed that the expression tendency of these genes was consistent compared to MOCK plants,indicating that GhPUB17 might function as a maverick which independent of SA and JA hormone immune pathways.4.GhCyP3 was identified as an interaction protein of GhPUB17 by related protein interaction assays.The cotton seedlings with GhCyP3 repressed by VIGS showed compromise to V.dahliae,suggesting its positive role in cotton resistance to V.dahliae.Furthermore,GhCyP3 was found as a repressor of E3 ligase activity of GhPUB17.Simultaneously,the antifungal activity of GhCyP3 was identified which could inhibit proliferation of V991 spores.Results above indicated that GhCyP3 has multiple controlling strategies on cotton resistance to V.dahliae.5.In order to make detailed functional analysis of PUB gene family,we made a genome-wide search of PUB genes using cotton genome sequence.To do this,the PUB genes in Arabidopsis thaliana were taken as references for PUB gene searching in Gossypium hirsutum,77 PUB genes were found and classified into 14 groups.The transcriptome data of cotton tissue expression and V991 inoculation were analyzed and the PUB genes were subsequently found expressing in different tissues and being induced at multiple time points after V991 inoculation.124 from 267 resequencing data from cotton cultivated varieties were used for SNP and InDel analysis in PUB genes,and the data of resistance performance to V.dahliae of which were simultaneously surveyed.We found that parts of PUB genes have SNPs(Single Nucleotide Polymorphisms)and InDels(insertion-deletion)inside the coding region and promoter area,and some of them might have possible relations to cotton resistance to V.dahliae.All results displayed above indicated that GhPUB17 functions as a negative regulator in cotton resistance to V.dahliae,while GhCyP3 positively regulates cotton resistance to V.dahliae,with repression behavior to E3 ligase activity of GhPUB17 and antifungal activity.Furthermore,the other members of PUB gene family might also involved in contton resistance to V.dahliae.These results could provide significant references for performing cotton molecular disease-resistance breeding.